EP1627073A2 - Use of a biotinylated polypeptide for determining the activity of protein-phosphorylating enzymes - Google Patents

Abstract

The invention relates to the use of a polypeptide for determining the ability of an enzyme, a functional fragment or a derivative thereof, to modulate the phosphorylation status of the polypeptide, said invention being characterised in that the polypeptide is biotinylated.

Description

Use of a polypeptide

The invention relates to the use of a polypeptide to determine the ability of an enzyme to modulate the phosphorylation state of the polypeptide. Further aspects of the invention relate to a method for determining such an activity and to a method for identifying substances which modify this ability of the enzyme.

Insulin is a peptide hormone that affects a large number of growth and metabolic pathways by binding to the insulin receptor, thereby activating its intrinsic tyrosine kinase. This event leads to the phosphorylation of a variety of proteins that can bind to the insulin receptor (IR) on specific tyrosine residues. The family of insulin receptor substrate (IRS) proteins also belongs to the proteins phosphorylated in this way.

Insulin receptor substrate 1 (IRS-1) is a cellular protein which can be phosphorylated by a large number of protein kinases (tyrosine-specific or serine / threonine-specific protein kinases) on tyrosine and / or serine residues and or threonine residues. Depending on the enzyme, different tyrosine or serine / threonine residues will presumably be phosphorylated specifically. In addition to tyrosine kinases such as the insulin receptor (White 2002), the IGF-1 receptor (White 2002) or JAK 1/2 (Thirone et al. 1999), IRS-1 is also known to be produced by serine / threonine kinases such as kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH (2) - terminal kinase (JNK, Aguirre et al. 2000) Protein kinase A (Sun et al. 1991) , Mitogen activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997), AMP activated kinase (Jakobsen et al. 2001) or Phosphoinositoi 3 Kinase (Freund et al. 1995) .phosphorylated. IRS molecules are key molecules in the insulin signal transduction pathway and play a central role in the maintenance of cellular functions such as growth, survival and metabolism. Phosphorylated IRS proteins serve as "docking" proteins with a variety of docking sites for the insulin receptor and a complex network of intracellular signaling molecules with so-called Signal Recognition Complex (SRC) homology 2 domains (SH2 Domains). The activation of these Sh2 domain proteins activates certain signal cascades, which in turn leads to the activation of various effectors that lie further down in the signal cascade, which ultimately leads to the transmission of the insulin signal to a branched series of other intracellular signal cascades (for an overview, see White 2002). IRS belongs to a group of phosphoproteins from 160 to 185 kDA in size that serve as a substrate for the insulin receptor. Four members of the IRS family (IRS-1, IRS-2, IRS-3 and IRS-4) are known. They differ in tissue distribution, subcellular localization, development-specific expression, type of binding to the insulin receptor and the type of SH2 proteins with which they interact. The four members of the IRS family are very similar in their basic protein structure: All have an amino (N) -terminal Plextrin homology domain (PH domain) that binds to membrane phospholipids, a phosphotyrosine binding domain (PTB domain) that is directly connects carboxy (C) terminal to the PH domain and is involved in the detection of the Asp-Pro-Glu phosphotyrosine (NPEpY) sequence which beta-insulin receptor in the juxta-membrane region. Subunit is localized. Furthermore, they have a somewhat less conserved C-terminal part, which has various potential tyrosine phosphorylation motifs to which special SH2 domain-containing proteins can bind.

IRS-1 contains 21 _possible, tyrosine phosphorylation sites, some of which are located in Aminpsäuresequenzmotiven that can bind to the SH2 Domjänen proteins. IRS-1 also contains 30 potential serine / threonine phosphorylation sites in motifs that can be recognized by various kinases such as kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c- Jun NH (2) terminal kinase (JNK, Aguirre et al. 2000) Protein kinase A (Sun et al. 1991), Mitogen activated protein kinase (Mothe et al. 1996), Protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997), AMP activated kinase (Jakobsen et al. 2001) or phosphoinositol 3 kinase (PI3 Kinase, Freund et al. 1995). Inhibitory effects on the insulin receptor signaling pathway can be at least partly due to the recently discovered role of serine / threonine phosphorylation of IRS-1 explained, which is associated with a deterioration in the interaction with the insulin receptor and / or a reduction in the tyrosine phosphorylation of IRS-1 and / or a deterioration in the interaction with subsequent signal proteins which can bind to tyrosine phosphorylated IRS-1 5 (for For an overview, see White 2002). For various kinases, for example kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH (2) terminal kinase (JNK, Aguirre et al. 2000) protein kinase A (Sun et al. 1991), mitogen-activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993),

10 glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997) or phosphoinositol 3 kinase (Freund et al. 1995) have so far been demonstrated to directly phosphorylate IRS-1 in vitro. In any case, increased kinase activity in intact cells inhibited the activity of the insulin signal transduction pathway. Furthermore, the in vitro phosphorylation of IRS-1 on serine / threonine

15 residues in some studies are directly related to the reduced tyrosine phosphorylation by the insulin receptor (Le Marchand-Brustel 1999)). The sequences of IRS-1, 2, 3 and 4 are publicly available. The coding polynucleotide sequences and the associated protein sequences of these genes are under the numbers NM_005544 (IRS-1 hs), XM: 007095 (IRS-2 hs), NM: 032074

20 (IRS-3 rat), NM: 003604 (IRS-4 hs) available from the NCBI Nucleotide Database. NCBI is the National Center for Biotechnology Information (postal address: National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, MD 20894, USA; web address: www.ncbi.nhm.nih.gov). The cloning of the IRS-1 gene was described, inter alia, in Araki et al. 1993 and Siemeister et. al,

25 1996; the cloning of IRS-2 to 4 was carried out by Araki et al 1994, Lavan et al. 1997a and Lavan et al. 1997bbeschrieben.

In order to determine the ability and to measure the activity of different kinases to phosphorylate IRS-1, various methods are known in the prior art which are based either on radioactive detection methods (e.g. transmission of radioactive

30 labeled phosphate on the substrate) or non-radioactive detection methods. It is known to determine the phosphorylation of IRS-1 on full-length IRS-1 protein, fragments or peptides thereof, which still have at least one phosphorylation site, by a method in which incubation with radioactively labeled ATP and the kinase to be tested is carried out depending on the ability of the kinase to phosphorylate IRS-1, radioactive phosphate residues are transferred to IRS-1. This is followed by a chromatographic or electrophoretic separation of the IRS-1 and detection of the amount of phosphate transferred by flow scintillation or autoradiography (as described, for example, for the complete IRS-1 protein and glycogen synthase kinase 3 beta in Eldar-Finkelman et al. 1997, for a fragment of IRS -1 (amino acid 516 - 777) and insulin receptor, IGF-1 receptor or recombinant insulin receptor kinase in Siemeister et al. 1995 or an IRS-1 peptide (amino acid 601 - 616) with cell lysates which contain activated protein kinase from the PKC family in De Fea et al. 1997. Furthermore, Siemeister et al. 1995 demonstrated the ability to phosphorylate IRS-1 fragments, for example a fragment of IRS-1 (amino acid 516-777) and insulin receptor, IGF-1 receptor or recombinant insulin receptor kinase Incubation with radioactively labeled ATP, dropping of the substrate onto a positively charged membrane (nitrocellulose or similar material), Washing and detection of the bound radioactively labeled substrate by means of autoradiography or measurement of the radioactive radiation.

Incubation of a biotinylated IRS-1 peptide (amino acid 601 - 616) with radioactively labeled ATP, dropping the substrate onto a streptavidin-coated membrane, washing and detection of the bound radioactively labeled substrate by autoradiography or measurement of the radioactive radiation is another method, to determine the ability of kinases to phosphorylate IRS-1 (see De Fea et al. 1997).

The disadvantage of the radioactive test methods described above is obvious, since the use of radioactivity entails considerable dangers, is very cost-intensive and is therefore not particularly suitable for high-throughput methods (HTS methods). The disadvantage of the methods described above, which are based on the use of short peptides, is that these peptides have unfavorable kinetic constants (Vmax, Km) and, moreover, the spatial structure in the case of peptides is very different from that of the physiological enzyme substrates. On the one hand, this manifests itself in a completely different folding, so that certain biological spaces that make up the specificity of the enzyme-substrate interaction are not present, which is either a lack of recognition (and thus modification) or an unspecific recognition (and thus modification) ) results and ultimately produces incorrect results. In addition, because of their brevity, peptides have only one or a few phosphorylation sites, so that different peptides are required for the investigation of the phosphorylation modification of a particular substrate by different enzymes. This also results in increased costs and a limited applicability for processes in HTS format. The object of the invention is therefore to provide a possibility for determining the activity of protein phosphorylating and / or dephosphorylating enzymes which does not have the disadvantages mentioned above. This object is achieved by using a polypeptide (Def GGs to the peptide) to determine the ability of an enzyme, a functional fragment or derivative thereof to modulate the phosphorylation status of a polypeptide, characterized in that the polypeptide is biotinylated. The invention is based on the results of the inventors, who surprisingly showed that even with polypeptides or full-length proteins there was no steric hindrance to the binding of biotin by streptavidin and that the biotinylation did not interfere with the phosphorylation of the substrate under investigation by kinases.

In the context of the present invention, the term polypeptide means a molecule containing amino acids linked by peptide bonds, which has at least 50 amino acids linked linearly in this way. Shorter molecules of this type are called peptides. The term protein refers to molecules that comprise at least one polypeptide chain, but can also consist of several polypeptide chains that are associated or linked with one another. The term protein thus includes the term polypeptide. According to a preferred embodiment of the various aspects of the present invention, the polypeptide has a length of 50 amino acids and more, preferably 50-300.

According to a further preferred embodiment of the various aspects of the invention, the polypeptide has a size of 1 kda and more, preferably 1 to 100 kDa and particularly preferably 10 to 50 kDa.

In the present case, the term substrate of an enzyme is understood to mean any molecule that is suitable for being modified by the enzyme. In the context of the present invention, natural substrates are molecules which are designed in such a way as they occur in nature in a physiological or pathological context and are capable of being modified by the enzyme in question.

The modulation of the state of phosphorylation by the enzyme denotes the type of modification of a substrate by an enzyme in which at least one phosphate group is transferred to or removed from the substrate. The enzymes relevant to the present invention therefore have the ability to catalyze one and / or the other reaction. They therefore have at least this ability of the kinases and / or the phosphatases, but can also have other enzymatic properties (e.g. protease properties, etc.). The various enzyme categories and their properties are well known to the person skilled in the art.

In the present case, a functional fragment of an enzyme is any fragment of the enzyme (that is, a molecule that is reduced or shortened compared to the naturally occurring form) that still has the ability to modulate the phosphorylation state of at least one polypeptide. The term "functional derivative" of an enzyme encompasses any type of modification of the enzyme compared to the naturally occurring form, which does not constitute a shortening, the derivative of the enzyme still having the ability to modulate the phosphorylation state of at least one polypeptide. The present invention also refers to functional derivatives of fragments of enzymes that can modulate the phosphorylation state of at least one polypeptide. The ability of the enzyme to modulate the phosphorylation state of the polypeptide can be determined both qualitatively and quantitatively (ie as a quantifiable measurement).

The use according to the invention has the advantage that the results obtained in this way are more meaningful due to the length of the substrates used, since they assume a tertiary structure that more closely corresponds to the physiological conditions. In addition, in contrast to the peptides known in the prior art, the polypeptides used have good kinetic constants (for example with IRS-1: Km 19 μM: compared to peptides:> 200 μM see Siemeister et al. 1995) and it is in the analysis of substrates with several phosphorylation sites only one substrate is necessary with which, for example the ability of various enzymes can also be determined.

A preferred embodiment relates to a use in which the ability of an enzyme to phosphorylate the polypeptide is determined. Particularly useful types of enzymes with kinase activity for the various aspects of the present invention relate to serine / threonine or tyrosine kinases. Particularly suitable examples of kinases include u. a. the insulin receptor, IGF-1 receptor, trK receptor, EGF receptor, casein kinase II, members of the protein kinase C family, protein kinase B / Akt, mitogen activated protein kinase (MAP kinase), GSK-3 beta, ERK1 / 2 , IKK beta kinase, AMP kinase, PI3 kinase or JNK. In addition, the use according to the invention is also suitable for determining the ability of an enzyme to dephosphorylate the polypeptide. Another aspect of the invention relates to a method for determining the ability of an enzyme, a functional fragment or derivative thereof to modulate the phosphorylation status of a biotinylated polypeptide.

Suitable methods for determining the degree of phosphorylation of biotinylated polypeptides relate, for example, to methods which are known to be suitable for determining the degree of phosphorylation of short peptides. These are familiar to the person skilled in the art. According to a preferred embodiment, the method according to the invention relates to a method in which the ability of an enzyme, a functional fragment or derivative thereof to phosphorylate the polypeptide is determined by the following steps: a) contacting the enzyme or functional fragment or derivative with the biotinylated polypeptide and starting the phosphorylation reaction in a suitable reaction mixture, b) contacting the reaction mixture with an a carrier-coupled agent capable of binding to the biotinylated polypeptide, c) determining the phosphorylation state of the polypeptide bound to the agent. A further preferred embodiment of the invention relates to a method for determining the ability of an enzyme, a functional fragment or derivative thereof to dephosphorylate the polypeptide with the steps a) bringing the enzyme or functional fragment or derivative into contact with the biotinylated polypeptide, which has at least one Has phosphate residue and start the phosphoryiation reaction in a suitable

Reaction batch, b) contacting the reaction batch with an agent coupled to a carrier capable of binding the biotinylated polypeptide, c) determining the phosphorylation state of the polypeptide bound to the agent.

The agent can be any type of molecule or supramolecular assembly (e.g. body or device) that is suitable for binding the biotinylated polypeptide. The binding can take place to the biotin portion or to the polypeptide itself, with one binding to the polypeptide itself

Binding dependent on the phosphorylation state is preferred (eg binding only in the phosphorylated or unphosphorylated state with reference to individual or more phosphorylation sites). Preferred embodiments of the agent therefore relate to streptavidin or phosphospecific antibodies (ie antibodies which recognize the phosphorylation of certain residues on the polypeptide and can bind specifically to the polypeptide phosphorylated there). The reaction mixture used in the various aspects of the invention can be biochemical (ie in vitro) or cellular. The composition of biochemical approaches depends on the requirements of the enzyme to be investigated, but suitable constituents and compositions, for example ATP, a buffer for setting a desired pH environment and a desired salt concentration for ensuring the enzyme activity are known to the person skilled in the art. In the case of biochemical approaches, enzyme and or polypeptide can be present recombinantly and / or as a molecule partially or completely purified from natural sources and / or in the form of extracts from biological material, in particular cell or tissue extracts.

Biological material can include, among others: the cells of a tissue or organ (e.g. brain, blood, liver, spleen, kidney, heart, blood vessels), preferably those of a vertebrate including humans, or cells from a cell culture. Cells used in the context of the invention include all types of cells, e.g. eukaryotic or prokaryotic unicellular organisms (such as bacteria, e.g. E. coli or yeasts, e.g. S. pombe or s. cerevisiae) or cell lines derived from multicellular organisms (such as HeLA, COS, NIH-3T3, CHO, etc.) , Mammalian cell lines are preferred. Cells from a tissue association or organ of a vertebrate, including humans, can be obtained using common techniques such as “blood collection, tissue puncture or surgical techniques. The production of such recombinant molecules, the purification of naturally occurring molecules from cells or tissues and the production of cell or tissue extracts is well known to the person skilled in the art (see also examples of the standard literature listed below).

Cellular systems suitable for use in the various aspects are also known to the person skilled in the art and preferably comprise isolated cells which originally come from tissue associations (preferably from vertebrates, particularly preferably mammals and in particular humans), particularly preferably in the form of cultivated cell lines; they also include unicellular organisms (eukaryotes or prokaryotes), such as yeast or bacterial cells, especially in the form of cultivated strains. Carriers can be all types of molecules or supramolecular assemblies (eg bodies or devices) which are suitable for removing or labeling the peptide coupled to them via the biotin-streptavidin interaction from the reaction mixture. Suitable devices are, for example, membranes, plates or bodies of various shapes (generally referred to as beads in the present case), made of various materials which are well known in the prior art. The type of carrier depends on the process objective (e.g. diagnostic, drug discovery or discovery of new interaction partners) and the type of detection, the selection of suitable carriers is within the range of expert knowledge.

According to one embodiment of the method according to the invention, radioactively labeled γ32P-ATP is added to the reaction mixture, and the phosphorylation state is determined by measuring the radioactivity remaining on the support, preferably the membrane or plate, after carrying out at least one washing step. In this way, the components of the reaction mixture, including free radioactivity, which are not bound to the streptavidin can simply be removed, so that the phosphorylation state of the polypeptide can easily be determined on the basis of the radioactivity immobilized on the support. Streptavidin is particularly suitable in this case as a means of binding the biotinylated polypeptide. According to a further preferred embodiment of the invention

In the method, an antibody (BSP) is added to the reaction mixture, which is able to bind specifically to the phosphorylated polypeptide. The antibody can both be the agent itself and can be added in addition to the agent (in which case it is then preferably not a phosphospecific antibody and particularly preferably streptavidin). The phosphorylation state is determined here by determining the amount of the antibody bound to the polypeptide. Suitable measures for labeling and detection of the antibody are known to the person skilled in the art. On the one hand, appropriately labeled first antibodies can be used that are directly detectable, or appropriately labeled second antibodies directed against the FC (crystalizing fragment) portion of the first antibody can be used, which increases the specificity of the detection. The term antibody encompasses both monoclonal antibodies and polyclonal antisera, recombinantly produced antibodies and recombinantly produced single-chain antibodies. The selection and production of such antibodies is within the range of expert knowledge, in this regard reference is also made to the standard literature listed below. Suitable labels for such antibodies are also known in the prior art and include, for example, enzymatic labels such as CIP (Calf intestinal phosphatase) or HRP (Horseraddish Peroxidase), fluorescent molecules which, when excited by irradiation with light of a certain wavelength, generate a detectable signal such as Texas Red, Cy3, FITC (fluorescein isothiocyanate), or known fluorescent proteins. The selection of suitable markings also corresponds to the professional ability. Suitable labeled or unlabeled first and second antibodies and their production are known in the art, moreover such antibodies are commercially available from various suppliers. First and second antibodies are available, for example, from Becton Dickinson, Pharmacia or Santa Cruz Biotech. According to a preferred embodiment of the above method, the amount of the antibody bound to the polypeptide is determined by determining the amount of the antibody remaining on the support, preferably the membrane or plate, after carrying out at least one washing step. According to a further preferred embodiment of the invention

Method, the carrier coupled to the agent is a first carrier that includes a first signal generator and the polypeptide is coupled to a second carrier that includes a second signal generator, the two signal generators being able to generate a detectable signal when they are are in close proximity to each other and the determination of the phosphorylation state using the

A determination is made as to whether a detectable signal has been generated. The carriers are preferably beads. The agent here is preferably a phosphospecific antibody. The carrier can be directly or indirectly connected to the antibody, preferably indirectly through ProteinA, which is coupled to the carrier. The second carrier can be bound directly or indirectly to the polypeptide, preferably indirectly through the biotin portion of the biotinylated polypeptide; this is preferably done via streptavidin coupled to the carrier. A signal generator can be any type of agent or molecule that is suitable for generating detectable signals; Examples include fluorophores that emit light upon excitation by energy, which can be detected directly or after signal amplification by suitable means known in the art. In the context of the present invention, the signal generators are selected such that a signal is only generated when the agent (ie preferably the phosphospecific antibody) interacts directly with the polypeptide. Suitable carriers and signal generators (for example in the form of the ALPHAScreen ™ or LANCE ™, Perkin-Elmer Life Sciences; HTRF ™, CIS Bio International) are known. The immediate proximity of the carriers to each other is crucial for signal generation. It is therefore very surprising that this type of method is suitable for use in conjunction with polypeptides, although these are significantly larger than the peptides used in the prior art. In the context of the various objects of the present invention, the polypeptide is preferably the natural substrate of the enzyme, preferably in an unabridged length.

Particularly suitable polypeptides include all substrates of the insulin receptor kinase. Particularly preferred polypeptides are insulin receptor substrate ^'(IRS) family, preferably IRS-1, 2, 3 or 4 and particularly preferably IRS-1, or functional fragments or derivatives thereof. That is fragments or derivatives (or derivatives of fragments) which have the ability to be phosphorylated by the insulin receptor. It is further preferred if the IRS is human IRS. Furthermore, the use of IRS-1, in particular human IRS-1 with the sequence according to SEQ ID No.1, is particularly preferred in the context of the various aspects of the present invention, human IRS-1 encoded by the sequence according to SEQ ID No.2. The aforementioned polypeptides are particularly suitable for determining the ability of the insulin receptor to phosphorylate them. A preferred IRS-1 fragment is a polypeptide with the amino acid sequence according to SEQ ID No.3. The various aspects of the invention can be used at different levels. Their use is particularly useful in the identification of Substances that modify the ability of the enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of the polypeptide. Suitable analytical methods or systems, so-called assays, which measure the activity or the concentration or amount or specific target molecules of the body (so-called “targets”, in this case the phosphorylation state of the polypeptide), as parameters of the effectiveness of potential active substances, are in the prior art These can be, for example, in vitro assays, that is to say biochemical assays with isolated or partially isolated components, which are put together to form a reaction mixture, by means of which the effectiveness of potential active substances can be measured. In addition, these can also be cellular test systems (assays) , in which the activity of the target protein (in this case the enzyme) and the effectiveness of potential active substances on the activity of this target molecule in the cellular environment can be determined.

An assay is any type of analytical method that can be used to monitor a biological process. Molecular processes and signal cascades, which represent parts of physiological metabolic pathways and control mechanisms, but also pathological states, are conventionally simulated in cellular or biochemical systems. The pharmacological activity of an active substance can then be determined on the basis of its ability to intervene in these pathways and mechanisms.

For use in the context of drug binding, in particular high-throughput screening for drugs, the assay must be reproducible and is preferably also scalable and robust (i.e. not very sensitive to external influences). The assay should preferably be suitable for high throughput screening of chemical substances for their ability to affect the activity of target molecules. The type of assay depends, among other things, on the type of target molecule used (eg exact type or type of basic biochemical molecule, eg polypeptide or polynucleotide) and the "read out", ie the parameters on the basis of which the activity of the target molecule is determined, from. Various types of assays are known in the prior art and for the most part are also commercially available from commercial suppliers.

Assays suitable for measuring the interaction of two binding partners include, for example, radioisotopic or fluorescent assays, for example fluorescence polarization assays, such as those commercially available from Panvera, Perkin-Elmer Life Sciences (NEN, LANCE ™, AlphaScreen ™) or CIS Bio International (HTRF ™). Other examples of assays include cellular assays in which a cell line expresses stably (inducible or constitutive; chromosomal or episomal) or transiently a recombinant protein as desired. These assays include, for example, reporter gene assays in which the regulation of a specific promoter or the regulation of a signal transduction path or a member of a signal transduction cascade is measured on the basis of the activity of a reporter enzyme whose expression is under the control of the promoter in question. For this type of assay it is necessary to generate a recombinant cell line which expresses the reporter gene under the control of a defined promoter which is itself under investigation or which is regulated by the signal transduction cascade to be examined. Suitable reporter enzymes are generally known to the person skilled in the art and include fireflies luciferase, Renilla luciferase (both commercially available, for example, from Packard Reagents), β-galactosidase, etc. The selection of suitable cell lines is known to the person skilled in the art and depends, inter alia, on the aim of the assay or read out ". These are usually cell lines that are easy to cultivate and transfect, such as HeLA, COS, CHO or NIH-3T3 cells. For example, fluorescence polarization, for example, are suitable for measuring protein phosphorylation or kinase activity Commercially available through Panvera, Homogeneous Time Resolved Fluorescence (HTRF ™, Cis Bio International) or LANCE ™ Assays (Perkin-Elmer Life Sciences) or the Amplified Luminescent Proximity Homogeneous Assay (ALPHAScreen ™ from Perkin-Elmer Life Sciences) present invention particularly expedient measurement of kinase activity using ALPHAScreen ™ from Perkin-Elmer Li fe sciences is carried out, for example, in that the kinase to be investigated phosphorylates a biotinylated peptide in a biochemical approach in the presence of ATP. The phosphorylated Peptide is then bound by a specific anti-phospho antibody to which protein A-conjugated acceptor beads or with suitable second antibodies are coupled. In the same approach are streptavidin-coupled donor beads that bind the biotin portion of the peptide. By binding to the peptide, acceptor and donor beads come in close proximity, which sets in motion a cascade of chemical reactions that generate a highly amplified, detectable luminescence signal: Laser excitation stimulates a photosensitizer in the donor bead to put ambient oxygen into a singlet status , The singlet oxygen then diffuses to the acceptor bead, where it excites a thioxane derivative, which emits chemiluminescence with a wavelength of 370 nm, which in turn stimulates further fluorophores in the acceptor bead to luminesce light with wavelengths from 520 to 620 nm. Since the singlet oxygen excites the fluorophores only when the donor and acceptor beads are in close proximity, detectable signals are only generated. Other types of assays and other types of "read outs" are also well known to the person skilled in the art.

The use in the form of high-throughput methods (HTS, High Throughput Screen) is particularly preferred, by means of which a large number of substances can be analyzed in a very short time. Depending on the objective, the modification of the modulation can mean an inhibition or activation of the modulation by the enzyme. The type of modification includes all possible influences that ultimately have an effect on the enzyme-catalyzed phosphorylation state of the polypeptide, such as the modification of the enzyme-substrate interaction or the modification of the catalytic activity of the enzyme, but also (preferably in the analysis by means of cellular reaction approaches ) modification of enzyme expression, etc.

Another aspect of the invention relates to a method for identifying substances which modify the ability of an enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of a polypeptide with the steps a) determining the ability of the enzyme or functional fragment or derivative from modulating the phosphorylation state of the polypeptide according to one of the above-mentioned methods according to the invention, the substance to be tested not being added to the reaction mixture, b) determining the ability of the enzyme or functional fragment or. Derivative of modulating the phosphorylation state of the polypeptide according to one of the methods according to the invention described above, the substance to be tested being added to the reaction mixture, c) comparing the ability according to a) with that according to b).

The methods according to the invention are particularly suitable for the identification of pharmacologically active substances for the treatment of non-insulin-dependent diabetes mellitus (NIDDM), in oncology (IGFRK) or for the treatment of inflammatory processes (IKK kinase).

The invention is explained in more detail below with the aid of various figures and examples, without thereby restricting the subject matter of the invention.

Example 1 :

IRS-1 fragment used

For the demonstration of the possibility of biotinylating a polypeptide substrate from the insulin signaling pathway and using it for the uses and methods according to the invention, a 262 amino acid fragment from human IRS-1 (aa516-aa777) was selected, which contains central potential tyrosine (fat ) as well as serine phosphorylation sites (underlined) (Siemeister et al.

J. Biol. 1995). The fragment contains five potential tyrosine phosphorylation sites, which are highlighted in FIG. 3 and subsequently together with their

Motifs are shown in bold.

516 -

DLDNRFRKRT HSAGTSPTIT HQKTPSQSSV ASIEEYTE M PAYPPGGGSG

GRLPGHRHSA FVPTRSYPEE GLEMHPLERR ^" GGHHRPDSST LHTDDGY P SPGVAPVPSG RKGSGDY P SPKSVSAPQQ IINPIRRHPQ RVDPNGYM M

SPSGGCSPDI GGGPSSSSSS SNAVPSGTSY GKLWTNGVGG HHSHVLPHPK PPVESSGGKL LPCTGDYMNM SPVGDSNTSS PSDCYYGPED PQHKPVLSYY SLPRSFKHTQ RP -777

Serins 612, 632, 662 and 731, which represent four possible serine kinase phosphorylation sites in YMXMSP motifs, are located near the tyrosine phosphorylation sites of the insulin receptor, which are housed in binding sites for SH2 somenas. The mutation of these serine residues to alanine leads to an increase in the IRS-1 -mediated activity of the phosphatidyl-insositol trisphosphate kinase (PI3K), (Mothe et al. 1996), which indicates that they have an inhibitory function. However, it cannot be ruled out that there are other serine phosphorylation sites that are not yet known. Example 2:

Cloning and biotinylation of hlRS-1-p30

For the investigation, the 262 amino acid domain D516-P777 (HLRS-1- p30) of human IRS-1 was first expressed in E-coli as described in Siemeister et al., 1995. The expression vectors were thereby inserted by inserting the polynucleotide with the sequence according to SEQ ID No. 10 (cDNA sequence of HLRS-1-p30) into the plasmid pET3d (commercially available under order number 69421 from Novagen), prepared by conventional methods. For this purpose, the empty vector was first digested with the enzymes Ncol (commercially available from Röche Diagnostics GmbH Mannheim under order number 835315) and BamHI (commercially available from Röche Diagnostics GmbH Mannheim under order number 656275) under standard conditions and using spin columns (commercially available from Qiagen, Hilden cleaned under order number 28104).

The biotinylation was carried out according to the order by the commercial provider N-Zyme, Darmstadt, Germany using common techniques. The expression of the HLRS-1-p30 insulin receptor fragments was carried out as described in Siemeister et al., 1995. To check the expression result, protein extracts from E. coli (strain: E. coli BL21, commercially available from Novagen under order number 69451-3 ), separated by SDS-PAGE under standard conditions (see, for example, the standard literature listed below) and stained with Coomassie staining solution according to standard conditions (see, for example, below) listed standard. Literature). The purification of HLRS-1-p30 was also carried out according to Siemeister et. al. The biotinylation of hlRS-1-p30 was carried out enzymatically using transglutaminase. Example 3 ALPHAScreen ™: Phosphorylation of biotinylated IRS-1 fragment by wheat germ lectin affinity-purified insulin receptor from rat liver. In the experiment, the result of which is shown in FIG. 4, wheat germ lectin affinity chromatographically purified insulin receptor from rat liver (WGA-IR, SEQACC number NP_058767 or commercially available from Sigma under order number 70543) with various concentrations of human insulin (for example commercially available from Sigma under order number 14) 266) and 85 nM biotinylated IRS fragment for 10 minutes at 4 ° C in 50 mM Tris buffer, pH 7.4, 8 mM MgCl2, 2 mM MnCl2, followed by a 30 minute incubation after addition of ATP (final concentration 50μM) at 30 ° C. The reaction was then stopped by adding EDTA to a final concentration of 20 mM and the phosphorylation of IRS-1 was detected by using a p-Tyr-specific antibody directly coupled to the acceptor (commercially available from Perkin-Elmer Life Sciences under order number 6760601 C), which resulted in the readout shown in FIG. With this method, the EC50 for insulin could be determined to be 10 nM.

Example 4

ALPHAScreen ™: Phosphorylation of biotinylated IRS-1 fragment by PKC and recombinant insulin receptor kinase.

Perkin-Elmer Life Sciences' ALPHAScreen ™ enables detection of the interaction between the phosphorylated IRS-1 fragment and antibodies that recognize phosphorylated serine or tyrosine residues (p-Ser / p-Tyr antibody). The biotinylated IRS-1 is bound to the streptavidin donor and the antibody by acceptor-coupled protein A or a suitable second antibody bound to the acceptor. When an interaction takes place, the acceptor gets and stays in the immediate vicinity of the donor, so that singlet oxygen atoms, which are generated by the donor can reach chemiluninescent groups in the acceptor bead by diffusion, which ultimately results in the emission of detectable light.

The light intensities (the so-called “readout”) generated in the aforementioned assay in the form of bar graphs in FIGS. 5 A and B were obtained after 30 minutes incubation of IRS-1 with protein kinase C and ATP and subsequent addition of p-Ser antibodies (commercial available from Biosource, Belgium under order number 44-550) and further incubation for 120 minutes detected and quantified by measurement with a Perkin-Elmer Fusion or AlphaQuest instrument.The comparison of the light intensities generated in the presence and absence of PKC is shown in Figure 11A. In the experiment, the result of which is shown in FIG. 11B, recombinant insulin receptor kinase (IRK, amino acid 941- (1343, NCBI accession number NM_000208) was incubated with polyiysin for 10 minutes at 30 ° C. in 50 mM Tris buffer, pH 7 , 4, 8 mM MgCl _2, 50μM ATP reaction buffer activated and then the IRK substrate IRS added, followed by a 30 minute incubation ion at 30 ° C. Phosphorylation of IRS-1 was detected using a p-Tyr specific antibody directly coupled to the acceptor (commercially available from Perkin-Elmer Life Sciences under order number 6760601 C), resulting in the readout shown in Figure 11B.

The aforementioned studies were able to demonstrate for the first time that ^" biotinylated polypeptides can be phosphorylated by kinases. This was demonstrated using a 28 kDA fragment of hlRS-1, which in the biotinylated state can be phosphorylated by the serine kinase PKCδ and by the tyrosine kinase of the insulin receptor Detection by phosphospecific antibodies was also successful without a steric hindrance due to the size of the polypeptide in connection with the biotin residue, which interfered with the detection reaction, thereby generating a homogeneous assay system based on the principle of the ALPHAScreens ™ The phosphorylation state of polypeptides can be determined using the purification and detection techniques possible through biotinylation.This assay principle was applied here for the first time to a protein fragment the size of a polypeptide (more precisely 28kDa) enables the improved search for pharmacologically active substances which interact with the phosphorylation and dephosphorylation machinery of the cell / the diagnosis of phosphorylation-dependent diseases / the identification of new protein kinases for special polypeptides on large, even structurally intact physiological substrates, which is the specificity of these studies underlying phosphorylations or dephosphorylations and thus significantly increases the meaningfulness of the data generated in this way. In the assay system used here, the readout was also non-radioactive but luminescent, which is an advantage for use in high throughput screening (HTS) procedures. The assay shown here can thus be used for the HTS of all enzymes modulating the phosphorylation status of polypeptides and proteins, such as kinases and phosphatases, for identifying novel active substances or for verifying known active substances. It is also suitable for other processes, such as the aforementioned processes for the search for new enzymes which phosphorylate certain polypeptides, for example new IRS-1 phosphorylating kinases in whole cell lysates.

Figure description Figure 1

Protein sequence from IRS 1 - IRS 4 (SEQ ID No. 1 to 4). The sequence access numbers (NCBI Protein Database) of the four family members are NM_005544 (IRS-1 hs),: M: 007095 (IRS-2 hs), NM: 032074 (IRS-3 hs), NM_003604 (IRS-4 hs).

Figure 2

Coding DNA sequence from IRS 1 - IRS 4 (SEQ ID No. 5 to 8). The sequence access numbers (NCBI nucleotide database of the four family members are NM_005544 (IRS-1 hs),: M: 007095 (IRS-2 hs), NM: 032074 (IRS-3 hs), NM_003604 (IRS-4 hs).

Figure 3 The 262 amino acid domain of the IRS-1 protein (hIRS-1-p30), which was used for the present studies. Serins 612, 632, 662 and 731 are underlined. YXXM tyrosine phosphorylation motifs are shown in bold.

FIG. 4 results of the ALPHAScreen using insulin receptor purified by wheat germ lectin affinity chromatography

Figure 5

Results of the ALPHAScreen ™

Figure 6

Overview of the interactions of insulin receptor, 1RS-1 and serine kinases

Figure 7 Overview of the possible molecular mechanisms of the inhibitory serine phosphorylation literature

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Current Protocols in Molecular Biology; regularly updated, e.g. Volume 2000; Wiley & Sons, Ine; Editors: Fred M. Ausubel, Roger Brent, Robert Eg. Kingston, David D. Moore, J.G. Seidman, John A. Smith, Kevin Struhl.

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Materials and methods Unless otherwise stated, the methods mentioned are standard methods known to the person skilled in the art and can be used, for example, in the above listed literature, in particular the literature on standard methods (also referred to in the present text as standard literature). Abbreviations

The three- or one-letter code was used for amino acids (generally also abbreviated as AA or AS) (see also the specified standard literature); The commonly used one-letter abbreviations were used for nucleotides (see also the given standard literature).

Claims

claims

1. Use of a polypeptide to determine the ability of an enzyme, a functional fragment or derivative thereof to modulate the phosphorylation status of the polypeptide, characterized in that the polypeptide is biotinylated.

2. Use according to claim 1 for determining the ability of an enzyme to phosphorylate the polypeptide.

3. Use according to claim 1 for determining the ability of an enzyme to dephosphorylate the polypeptide.

4. A method for determining the ability of an enzyme, a functional fragment or a derivative thereof to modulate the phosphorylation status of a biotinylated polypeptide.

5. The method according to claim 4 for determining the ability of an enzyme, a functional fragment or derivative thereof to phosphorylate the polypeptide with the steps a. Contacting the enzyme or functional fragment or derivative with the biotinylated polypeptide and starting the phosphoryiation reaction in a suitable reaction mixture, b. Contacting the reaction mixture with an agent coupled to a carrier that has the ability to bind to the biotinylated

Bind polypeptide, c. Determination of the phosphorylation state of the polypeptide bound to the agent.

6. The method according to claim 4 for determining the ability of an enzyme, a functional fragment or derivative thereof to dephosphorylate the polypeptide with the steps a. Contacting the enzyme or functional fragment or derivative with the biotinylated polypeptide which has at least one phosphate residue and starting the phosphorylation reaction in a suitable reaction mixture, b. Contacting the reaction mixture with a carrier-coupled agent that has the ability to bind to the biotinylated polypeptide, c. Determination of the phosphorylation state of the polypeptide bound to the agent.

7. The method according to any one of claims 4 to 6, characterized in that the carrier is a membrane or plate.

8. The method according to any one of claims 4 to 7, characterized in that the agent is streptavidin or a phosphospecific antibody.

9. The method according to claim 7 or 8, characterized in that the reaction mixture radioactively labeled γ32P-ATP is added and the determination of the phosphorylation state is carried out by measuring the radioactivity remaining on the membrane or plate after carrying out at least one washing step.

10.The method according to any one of claims 4 to 8, characterized in that an antibody is added to the reaction mixture which is capable of binding specifically to the phosphorylated polypeptide and the determination of

Phosphorylation state by determining the amount of the antibody bound to the polypeptide.

11. The method according to claim 10, characterized in that the determination of the amount of the antibody bound to the polypeptide by the

The amount of antibody remaining on the membrane or plate is determined after at least one washing step has been carried out.

12. The method according to claim 10, characterized in that the carrier coupled to the streptavidin is a first carrier which comprises a first signal generator and the polypeptide is coupled to a second carrier which comprises a second signal generator, the two signal generators being capable of generate a detectable signal when they are in close proximity

Are close to each other and the determination of the phosphorylation state is based on the determination of whether a signal has been generated.

13. Use according to one of claims 1 to 3 or method according to one of claims 4 to 12, characterized in that the polypeptide has a length of 50 amino acids and more, preferably 50 to 300.

14. Use according to one of claims 1 to 3 or method according to one of claims 4 to 12, characterized in that the polypeptide is a

Size of 1 kda and more, preferably 1 to 100 kDa, particularly preferably 10 to 50 kDa and in particular 25 to 35 kDa.

15. Use according to one of claims 1 to 3 or 14 or method according to one of claims 4, 5 or 7 to 14, characterized in that the enzyme is a kinase, preferably a tyrosine kinase.

16. Use according to one of claims 1 to 3 or 12 to 15 or method according to any one of claims 4 to 15, characterized in that the polypeptide is the natural substrate of the enzyme, preferably in unabridged length.

17. Use according to one of claims 1 to 3 or 12 to 16 or method according to one of claims 4 to 16, characterized in that the polypeptide insulin receptor substrate (IRS), preferably 1RS-1, 2, 3 or 4 and particularly preferably IRS-1 or a functional fragment or derivative thereof.

18. Use or method according to claim 17, characterized in that the IRS-1 is human IRS-1 with the sequence according to SEQ ID No.1 or is encoded by the sequence according to SEQ ID No.2.

19. Use or method according to claim 18, characterized in that the IRS-1 fragment has the amino acid sequence according to SEQ ID No.3 and preferably consists thereof.

20. Use according to one of claims 1 to 3 or 12 to 19 or method according to one of claims 4 to 19, characterized in that the enzyme is one of the following kinases or a functional fragment or derivative thereof: insulin receptor, IGF-1 receptor, trK receptor, EGF receptor, casein kinase II, protein kinase C, protein kinase B / Akt, mitogen activated protein kinase (MAP kinase), GSK-3, ERK or JNK.

21. Use according to one of claims 1 to 3 or 12 to 20 or method according to one of claims 4 to 20 for the identification of substances which modify the ability of the enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of the polypeptide.

22. A method of identifying substances that modify the ability of an enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of a polypeptide with steps a. Determining the ability of the enzyme or functional fragment or derivative thereof to modulate the phosphorylation state of the polypeptide according to a method according to any one of claims 3 to 21, wherein the substance to be tested is not added to the reaction mixture, b. Determination of the ability of the enzyme or functional fragment or derivative thereof to increase the phosphorylation state of the polypeptide modulate according to a method according to any one of claims 3 to 21, wherein the substance to be tested is added to the reaction mixture, c. Comparison of the ability according to a) with that according to b).

23. Use according to claim 21 or method according to one of claims 21 or 22 for the identification of pharmacologically active substances for the treatment of non-insulin-dependent diabetes mellitus (NIDDM).